CN117546269A

CN117546269A - System and method for single ion mass spectrometry with time information

Info

Publication number: CN117546269A
Application number: CN202280044430.6A
Authority: CN
Inventors: D·M·斯坦; N·德拉克曼; B·威纳
Original assignee: Brown University
Current assignee: Brown University
Priority date: 2021-04-23
Filing date: 2022-04-22
Publication date: 2024-02-09
Also published as: WO2022226279A1; EP4327352A1; KR20240026444A; JP2024519194A

Abstract

The present disclosure relates generally to mass spectrometers, including but not limited to mass spectrometers capable of emitting ions at determinable times. In some aspects, the time between the time the ions leave the ion source and the time the ions reach the detector may be determined with relatively high temporal resolution, which may be useful for certain applications such as biopolymer sequencing. Further, in some cases, a relatively high number of ions exiting the ion source, such as at least 50% or more of the ions generated, may be determined at the detector. Other aspects generally relate to systems and methods of using such mass spectrometers, techniques involving such mass spectrometers, and the like.

Description

System and method for single ion mass spectrometry with time information

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional patent application Ser. No. 63/179046, titled "Systems and Methods for Single-Ion Mass Spectrometry with Temporal Information," filed by Stein et al at 2021, 4, 23, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates generally to mass spectrometers, including but not limited to mass spectrometers capable of emitting ions at determinable times.

Background

Mass spectrometry may be well suited for protein sequencing because it is an analytical technique that can recognize all 20 amino acids. Typically, mass spectrometers measure ions using a single ion detector and a mass filter that scans a narrow mass transmission window in time. For example, a quadrupole mass filter allows only ions within a narrow m/z range to pass, with a particular m/s range controlled by time-varying voltages applied to the four poles of the quadrupole mass filter. The ion transport rate is measured by a detector while scanning the allowed m/z range, and the mass spectrum is determined after at least one scan. Ions with m/z outside the transmission window do not pass through the filter. Thus, a particular ion leaving the ion source can only be detected and identified when it happens to pass through the filter at a time when the window is at the m/z center of the ion, which is not guaranteed if the order of the ion m/z values is not known in advance. Thus, such a system cannot be used for sequencing because it is not known when and in what order ions leave the ion source. Thus, mass spectrometers have not been able to be used for sequencing individual proteins.

Disclosure of Invention

The present disclosure relates generally to mass spectrometers, including but not limited to mass spectrometers capable of emitting ions at determinable times. In some cases, the subject matter of the present disclosure includes a variety of different uses for related products, alternative solutions to particular problems, and/or one or more systems and/or articles.

One aspect relates generally to mass spectrometers. In one set of embodiments, a mass spectrometer includes an ion source comprising a capillary and an electrode in proximity to the capillary, a magnetic mass filter downstream of the ion source, and a detector array downstream of the magnetic mass filter. In some cases, the capillary tube includes openings having a cross-sectional dimension less than 125 nm.

In another set of embodiments, a mass spectrometer comprises: an ion source constructed and arranged to generate individual ions or clusters of ions; a magnetic mass filter positioned to receive individual ions or clusters of ions from the ion source; a pump capable of generating a pressure of less than 100mPa in an environment between the ion source and the magnetic mass filter; and a detector array positioned to receive individual ions or clusters of ions from the magnetic mass filter.

In yet another set of embodiments, a mass spectrometer includes an ion source, a magnetic mass filter downstream of the ion source, and a detector array downstream of the magnetic mass filter.

Another aspect relates generally to a method of sequencing a biopolymer. According to one set of embodiments, the method includes ionizing a biopolymer contained within a fluid into ions or clusters of ions, passing the ions or clusters of ions through a magnetic mass filter, directing the ions or clusters of ions to a detector array, and determining a sequence of the biopolymer by determining the ions or clusters of ions with the detector array.

In another set of embodiments, the method includes delivering a fluid comprising a biopolymer into a capillary defining an opening, applying an electric field to ionize the biopolymer near the opening to produce ions or ion clusters, delivering the ions or ion clusters directly into an environment having a pressure of no more than 100mPa, passing the ions or ion clusters through a magnetic mass filter, directing the ions or ion clusters to a detector array, and determining a sequence of the biopolymer by determining the ions or ion clusters with the detector array.

Yet another aspect relates generally to a method of determining a concentration. In one set of embodiments, the method includes ionizing molecules from the fluid into ions or clusters of ions, passing the ions or clusters of ions through a magnetic mass filter, directing the ions or clusters of ions to a detector array, and determining the concentration of the molecules in the fluid by determining the ions or clusters of ions with the detector array.

Another aspect relates to a method comprising ionizing molecules in a fluid into ions or clusters of ions, passing at least 50% of the ions or clusters of ions through a magnetic mass filter, and directing the ions or clusters of ions to a detector.

Another aspect relates to a method comprising ionizing molecules from a fluid into ions or clusters of ions using an ion source, passing the ions or clusters of ions through a mass filter, directing the ions or clusters of ions to a detector, and determining a duration between a time the ions or clusters of ions leave the ion source and a time the ions or clusters of ions reach the detector.

Another aspect relates to a method comprising ionizing molecules using an ion source to produce ions or ion cluster sequences, passing the ions or ion cluster sequences through a mass filter, and directing the ions or ion cluster sequences to a detector array. In some cases, at least 50% of the ions or ion clusters that reach the detector array arrive sequentially. Furthermore, in some cases, at least 90% of the ions or ion clusters that reach the detector array arrive sequentially.

Other advantages and novel features of the disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the drawings.

Drawings

Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure nor is every component of every embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the drawings:

FIG. 1 illustrates a nano Kong Zhipu meter according to an embodiment;

FIGS. 2A-2B illustrate mass spectra of positive amino acid ions delivered directly into a high vacuum from a nanopore ion source in another embodiment;

FIG. 3 is a schematic diagram of a mass spectrometer according to yet another embodiment;

figures 4A-4D illustrate the operation of a nano Kong Zhipu meter according to certain embodiments;

FIGS. 5A-5C illustrate mass spectra of certain biomolecules according to some embodiments;

FIGS. 6A-6E illustrate the generation or emission of ions from an ion source in other embodiments; and

fig. 7 shows a magnetic mass filter according to yet another embodiment.

Detailed Description

The present disclosure relates generally to mass spectrometers, including but not limited to mass spectrometers capable of emitting ions at determinable times. In some aspects, the time between the time the ions leave the ion source and the time the ions reach the detector may be determined with relatively high temporal resolution, which may be useful for certain applications such as biopolymer sequencing. Further, in some cases, a relatively high number of ions exiting the ion source may be determined at the detector, such as at least 50% or more of the ions produced. Other aspects generally relate to systems and methods of using such mass spectrometers, techniques involving such mass spectrometers, and the like.

Conventional mass spectrometers typically use a single ion detector and a mass filter to measure ions, the mass filter scanning a narrow mass transmission window in time. For example, when the allowed mass-to-charge ratio range is swept, only ions having mass-to-charge ratios within the allowed range may be measured. Thus, conventional mass spectrometers can only determine the mass to charge ratio of a portion of the ions emitted. Furthermore, the capabilities of conventional mass spectrometers can be quite limited. For example, conventional mass spectrometers cannot be used to provide valuable information about ion sequencing, ion association, and/or ion sequence. Accordingly, certain aspects of the present disclosure are directed to mass spectrometers that can be used to provide such information.

In one example, a mass spectrometer includes a particular combination of components and/or configurations that impart enhanced detection and measurement capabilities to the mass spectrometer. For example, a mass spectrometer may include an ion source capable of ionizing molecules into individual ions, a magnetic mass filter capable of classifying ions based on their mass-to-charge ratios, and a detector array capable of determining the individual ions. The combination of these features may advantageously allow for the determination of the sequence, structure and/or identity of the species of interest (e.g., biopolymer). For example, mass spectrometers can be used to sequence proteins or nucleic acids. Advantageously, the mass spectrometers described herein can have high time resolution (e.g., less than 1 microsecond) and high overall ion transport efficiency (e.g., greater than or equal to about 0.8) compared to conventional mass spectrometers.

One non-limiting example of a mass spectrometer is shown in figure 1. In this example, ions (or ion clusters) are generated from an ion source, which in some cases is capable of generating ions at determinable times. The ion source may include a capillary tip that may allow direct ion evaporation of the sample with an applied electric field. In some cases, the tip may have an opening with a cross-section of less than 100 nm. Examples of such systems can be seen in PCT application entitled "Nanotip Ion Sources and Methods" filed on the same date as the present application and U.S. patent application serial No. 63/015407 entitled "Nanotip Ion Sources and Methods" filed on 24 th 4 th 2020, each of which is incorporated herein by reference in its entirety. The fluid enters the ion source via the fluid inlet and molecules within the fluid are converted into ions or clusters of ions in the ion source. In addition, ions (or clusters of ions) exiting the ion source may optionally pass through an ion lens or other suitable ion optics, for example, to focus the ions, and enter a chamber having a relatively low pressure environment ("vacuum" chamber), for example, a pressure of less than 100mPa (absolute). In some embodiments, individual ions or clusters of ions from the ion source may be emitted directly into a vacuum or low pressure environment. In some embodiments, the mass spectrometer includes a pump for creating such a vacuum or low pressure environment.

The ions may then be classified using a mass filter, for example, based on mass or mass to charge ratio. In some embodiments, a mass filter (e.g., a magnetic mass filter) may be downstream of the ion source. The mass filter may be positioned to receive individual ions or ion clusters from the ion source. One non-limiting example is a magnetic sector mass filter. In some cases, ions or ion clusters having different mass-to-charge ratios may deflect at different angles due to the magnetic field generated by the magnetic mass filter. Thus, ions of different mass or mass to charge ratios may have different trajectories when exiting the magnetic mass filter.

The ions or ion filters, upon exiting the magnetic mass filter, may be directed toward a detector array, for example downstream of the mass filter (e.g., magnetic mass filter). The detector array may be arranged and constructed to detect individual ions or clusters of ions passing from the mass filter. Because the ions do not necessarily have the same trajectories, a detector array may be used that is positioned to receive ions having different trajectories. Each detector may detect ions, for example, having a particular mass or mass to charge ratio with respect to time; the position of the detector is related to the incident mass or mass-to-charge ratio of ions arriving at the detector. Thus, since the time at which ions or ion clusters leave the ion source is known and there is no substantial collision (e.g., with air molecules) between the ion source and the detector array, the travel time between them can be determined, for example, with a relatively high time resolution (e.g., microseconds or less). In contrast, mass spectrometry systems that include air and/or time-varying voltages cannot achieve such time resolution or perform sequencing.

Thus, in some embodiments, a mass spectrometer as described herein may have a relatively high temporal resolution. In some embodiments, the mass spectrometer has a time resolution of less than or equal to 1 microsecond (less than or equal to 500 nanoseconds, less than or equal to 250 nanoseconds, less than or equal to 100 nanoseconds, less than or equal to 50 nanoseconds, less than or equal to 10 nanoseconds, etc.).

In some cases, information about ion travel time may be used for certain types of sequencing, e.g., monomers forming a polymer, such as amino acids or nucleotides in a protein or nucleic acid, may be sequentially ionized and transferred to a detector, and this information is used to reconstruct or "sequence" the original polymer. Fig. 3 shows another non-limiting embodiment of a mass spectrometer, for example for sequencing polymers, such as proteins in this example. As shown, the mass spectrometer includes an ion source (e.g., having nanopores) capable of emitting individual ions and ion clusters into a vacuum chamber, an electrode proximate the ion source, a magnetic mass filter downstream of the ion source, and a detector array (e.g., an individual ion detector) downstream of the magnetic mass filter.

Furthermore, in some embodiments, a relatively high number of ions or ion clusters generated by the ion source may pass through the magnetic mass filter to the detector, for example, due to the relatively low pressure present in the mass spectrometer. In some cases, at least 50% or more of the generated ions may reach one of the detectors. This can also be used, for example, to determine the concentration of certain ionizable molecules within the fluid.

Accordingly, certain aspects of the present disclosure relate to systems and methods related to mass spectrometers. In some embodiments, a mass spectrometer includes an ion source constructed and arranged to produce individual ions or clusters of ions. Ion clusters may be meant to include a single ion and multiple solvent molecules. For example, an ion cluster may include ions (e.g., water) having only 1 or 2 solvent molecules.

The ion source may be any of a variety of ion sources, such as an ion source capable of ionizing a species of interest (e.g., a biopolymer) into a single ion or cluster of ions. For example, in one set of embodiments, ion sources are described herein that include a capillary and an electrode in proximity to the capillary. The capillary tube includes openings having a cross-sectional dimension of less than 125nm (e.g., less than 100nm, less than 60nm, etc.). In some embodiments, the electrodes may be used to apply an electric field to the fluid within the capillary such that molecules from the species of interest in the fluid may be ionized into individual ions or clusters of ions. Specific configurations and components of the ion source are disclosed in more detail below.

For example, in some embodiments, the ion source may include a capillary and an electrode, which in some cases may be annular, between which a voltage is applied to generate ions. In some cases, the capillary tube may have an internal tip diameter of less than 125nm or less than 100 nm. This may allow ions to evaporate directly from the meniscus (meniscus) of the fluid in the capillary, bypassing the wasteful droplet evaporation process. In this state, ion evaporation may account for a substantial portion of the ion stream, and this emission pattern may be achieved with relatively low salinity solutions in some cases. In some embodiments, tips with an inner diameter less than 125nm or less than 100nm are capable of producing a high proportion of bare ions or ion clusters, e.g., including a small number of solvent molecules, e.g., only 1 or 2 solvent molecules. The small area of the liquid vacuum interface may in some cases prevent significant heat loss from evaporation, which allows for the use of volatile solvents such as water in some cases. Methods such as these may be used in some embodiments to analyze molecules or ions, e.g., biomolecules such as amino acids, nucleic acids, peptides or proteins, and the like. In some cases, ion sources such as those described herein can increase the sensitivity of mass spectrometry experiments, allowing single molecule protein sequencing or single cell proteomics research. Other applications are possible, such as described below.

For example, some embodiments generally relate to an ion source that includes a capillary and an electrode. The electrodes may be used to generate ionized molecules directly from the fluid within the capillary, e.g., into a reduced pressure environment or vacuum, e.g., at a pressure of 100mPa, or other pressures described herein. In some embodiments, the size of the capillary opening is such that when an electric field is applied, the fluid within the capillary forms a charged meniscus and species within the fluid exit the charged meniscus, for example by evaporation of predominantly ions. The use of capillaries with submicron openings (e.g., less than 100 nm) may facilitate fluid ionization by ion evaporation, where the species exiting the capillaries are ionized directly into single charged ions or clusters of charged ions, in contrast to electrospray ionization, where the species exiting the capillaries exit through a liquid jet that breaks into charged droplets that further break into charged ions in the presence of background gas, although it should be appreciated that some electrospray ionization may still occur in some cases. In some applications ion evaporation may be preferred, for example where efficient use or generation of individual ions from a fluid is required. For example, certain embodiments relate to ion sources in mass spectrometry where individual charged ions can be directly generated and subsequently detected.

According to one set of embodiments, the ion source includes a capillary defining an opening having a cross-sectional dimension (e.g., an inner diameter of the capillary) of less than 100nm. In some cases, the openings may also be sized such that ion evaporation dominates the liquid jet formation when the electric field is applied. For example, in certain embodiments, at least 50% of the exiting species may exit by ion evaporation or in the form of ions or ion clusters. For example, nanoscale capillaries can allow ions to evaporate directly from a fluid meniscus. In some embodiments, the fluid may be delivered into a capillary having such openings and delivered directly into a reduced pressure or vacuum environment (e.g., having a pressure of no more than 100 mPa) in the form of ions and ion clusters. Ions and ion clusters may be analyzed by mass filters and ion detectors in mass spectrometers or applied in other applications such as those described herein.

It should be appreciated that other types of ion sources may be employed. For example, the ion source may comprise a pulsed laser capable of ionizing molecules of the species of interest into individual ions or clusters of ions.

Some embodiments include ionizing molecules contained within a fluid into ions or clusters of ions. In some embodiments, the molecules may be ionized into individual ions or clusters of ions (i.e., individual ions clustered together with solvent molecules). In some embodiments, the molecules may be ionized into a single ion, with few, if any, ion clusters present.

In one set of embodiments, the molecules contained within the fluid may be molecules from a polymer or biopolymer (e.g., protein, polypeptide, nucleic acid, etc.). In another set of embodiments, the molecules may be small molecules (e.g., monomers, biomonomers, salt ions, etc.) contained within a fluid that are capable of being ionized from the fluid.

These molecules may be ionized into individual ions or clusters of ions by any suitable ion source. In one set of embodiments, the ion sources described herein may be used to ionize molecules from a fluid. For example, the ion source may comprise a capillary tube having a relatively small opening. In some embodiments, a fluid containing a species of interest (e.g., a biopolymer) may be delivered into the opening of the capillary. By applying an electric field to the fluid near the opening, molecules within the species of interest (e.g., biopolymer) can be ionized to produce individual ions or clusters of ions. Specific embodiments relating to such ion sources are described below.

While the above embodiments describe an ion source comprising a capillary, it should be appreciated that any type of ion source may be used in a mass spectrometer, provided that the ion source is capable of producing a single ion or ion cluster. For example, in one set of embodiments, the molecules may be ionized into individual ions or clusters of ions by a pulsed laser.

In some embodiments, a polymer, such as a biopolymer, contained within a fluid may be ionized into ions or clusters of ions. Examples of biopolymers include, but are not limited to, proteins, peptides, nucleic acids such as DNA or RNA, carbohydrates, polysaccharides, and the like. These can ionize into monomeric components such as amino acids, nucleotides, sugar units, monosaccharides, or the like. In some embodiments, upon ionization, individual ions or clusters of ions may be sequentially released from the biopolymer. For example, in some cases, a single ion may be released from a biopolymer at a time. Advantageously, the ability to ionize biopolymers into individual ions or clusters of ions and sequentially release them can reveal spatial and/or temporal information about the ordering or sequence of ions in the molecule. For example, the biopolymer may be ionized into a sequence of ions or clusters of ions (e.g., ionized monomers) that corresponds to a sequence of basic components (e.g., monomers) associated with the biopolymer prior to ionization.

Fig. 3 shows a non-limiting example of such an embodiment. As shown, an ion source (e.g., a nanopore) may be used to ionize a biopolymer (e.g., a protein) into individual ions (e.g., amino acids) or clusters of ions (e.g., amino acids with solvent molecules). Individual ions may be released from the biopolymer as ions or a sequence of ion clusters in a sequential order. As shown, the ion sequence released may correspond to the ion sequence in the biopolymer prior to ionization.

In addition, ions or ion clusters can be generated by the ion source at various rates. In some cases, ions or ion clusters may be advantageously generated at a relatively high rate. In some embodiments, the ions or ion clusters are generated at a rate greater than or equal to 1 (ion or ion cluster)/microsecond, greater than or equal to 5/microsecond, greater than or equal to 10/microsecond, greater than or equal to 25/microsecond, greater than or equal to 50/microsecond, or greater than or equal to 75/microsecond. In some embodiments, the ions or ion clusters are generated at a rate of less than or equal to 100 (ions or ion clusters)/microsecond, less than or equal to 75/microsecond, less than or equal to 50/microsecond, less than or equal to 25/microsecond, less than or equal to 10/microsecond, or less than or equal to 5/microsecond. Combinations of the above ranges are possible (e.g., greater than or equal to 1/microsecond, less than or equal to 100/microsecond). Other ranges are also possible.

In some cases, relatively high ionization rates may be useful for sequencing polymers (such as biopolymers). In some embodiments, for example, the biopolymer is a protein, e.g., comprising an amino acid sequence. In some embodiments, the protein may be ionized (i.e., produced) at any suitable rate within one or more of the ranges described above, e.g., to produce amino acids (or components thereof) that may be analyzed as described herein to determine the protein sequence. As another example, in some embodiments, the biopolymer is a nucleic acid, e.g., DNA, RNA, etc. In some embodiments, the nucleic acid may be ionized (i.e., produced) at any suitable rate within one or more of the ranges described above, e.g., to produce nucleotides or other nucleic acid fragments that may be analyzed as described herein to determine a nucleic acid sequence. For example, amino acids can ionize at a rate of at least 1 base per microsecond (at least 1 base per microsecond, at least 100 bases per microsecond, etc.).

After exiting the ion source, in some aspects, the ions or ion clusters are emitted directly into an environment (e.g., a vacuum chamber) having a relatively low pressure. The environment may have any of a variety of pressures or configurations, such as those described in detail below. For example, in some cases, the environment may be an environment having a pressure of no more than 100mPa (e.g., no more than 10mPa, no more than 1mPa, no more than 0.1mPa, etc.). In some embodiments, ions or ion clusters from the fluid are directed into a vacuum environment, such as from an ion source. Furthermore, it should be appreciated that the vacuum environment need not be a perfect vacuum.

In some aspects, the emitted ions or ion clusters may pass through a mass filter contained within a relatively low pressure environment, for example, as described herein. Furthermore, in some embodiments, the emitted ions may optionally pass through ion optics before passing through a mass filter, as described below.

In some embodiments, the mass filter is a magnetic mass filter. In some embodiments, the magnetic mass filter separates ions or ion clusters according to their mass-to-charge ratio by applying a magnetic field. The magnetic mass filter is capable of directing (e.g., bending) incident ions or ion clusters in different directions (e.g., angles) according to its mass-to-charge ratio. For example, as shown in fig. 3, incident ions and ion clusters having different mass-to-charge ratios may be directed or bent in different directions under the magnetic field generated by the magnetic mass filter. Furthermore, in some embodiments, a relatively large percentage of the emitted ions or ion clusters may pass through the magnetic mass filter. For example, at least 50% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or all) of the emitted ions or ion clusters can pass through the magnetic mass filter. Systems and methods for determining a relatively high percentage of ions generated by an ion source are discussed in more detail herein.

Various mass filters may be used. Non-limiting examples of mass filters include, but are not limited to, quadrupole mass filters, magnetic sector mass filters, and the like.

For example, as described above, in some embodiments, the mass filter may comprise a magnetic mass filter. In some embodiments, the magnetic mass filter includes a magnet and a yoke associated with (e.g., housing) the magnet. The magnetic mass filter may include a magnet formed from any of a variety of magnetic materials, including but not limited to rare earth elements, magnetic metallic elements, magnetic composite materials (e.g., ferrite), and the like. In one set of embodiments, the magnet is a permanent magnet comprising neodymium. In one set of embodiments, the yoke comprises iron.

In some embodiments, the magnetic field passes through an opening of the magnetic mass filter through which incident ions and ion clusters pass, and may be deflected to different angles by the magnetic field generated by the magnetic mass filter. For example, as shown in fig. 7, the magnetic mass filter has a central opening in which a magnetic field exists in the axial direction. As ions and ion clusters pass through the central opening, the ions or ion clusters are deflected by the magnetic field by different angles, for example, depending on their mass or mass-to-charge ratio.

The openings in the magnetic mass filter can have various sizes and shapes. In some cases, the opening may have a cylindrical, square, rectangular, or the like shape. The opening may have a first cross-sectional dimension (e.g., diameter, width, length) and a second cross-sectional dimension (e.g., height). In one set of embodiments, the first cross-sectional dimension (e.g., diameter) of the opening is greater than the second cross-sectional dimension (e.g., height). In some embodiments, the opening may have a first dimension (e.g., diameter) of at least 4cm (e.g., at least 5cm, at least 6cm, at least 8cm, at least 10cm, etc.). In some embodiments, the opening may have a second dimension (e.g., height) of at least 1cm (e.g., at least 2cm, at least 3cm, at least 4cm, at least 5cm, etc.). In some embodiments, the ratio of the first dimension to the second dimension of the opening is at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, etc.).

The magnetic mass filter can generate magnetic fields having various field strengths. In some embodiments, the magnetic field strength may be at least about 0.1T, at least 0.2T, at least 0.3T, at least about 0.5T, at least about 0.7T, at least about 1T, at least about 5T, etc.

In some embodiments, optionally, the ions and ion clusters may pass through an ion bender after exiting the mass filter. The ion bender may be configured to deflect ions and clusters of ions exiting the mass filter to the detector. For example, as a non-limiting example, ions or ion clusters are transferred from an ion bender to a detector. In some embodiments, a detector may be used to determine ions or ion clusters.

In some aspects, ions or clusters of ions passing through a mass filter (e.g., a magnetic mass filter) may be directed to one or more detectors, e.g., a detector array (e.g., a single ion detector), such as those described herein. For example, as shown in fig. 3, a magnetic mass filter directs and bends ions or ion clusters toward a detector array.

In some embodiments, ions or ion clusters having a particular mass or mass to charge ratio may be directed to respective detectors within the detector array, e.g., based on the amount of deflection that occurs as the ions or ion clusters pass through the mass filter. Ions or ion clusters having a larger charge amount may deflect to a greater extent than ions or ion clusters having a smaller charge amount. Thus, one or more detectors may be positioned to receive ions or ion clusters having different deflection amounts, which may then be used to determine the mass or mass-to-charge ratio of the incident ions or ion clusters. Thus, for example, the detector array may be positioned to determine the mass or mass-to-weight ratio of ions or ion clusters based on the impact locations of the various ions or ion clusters within the array on the various detectors. Further, in some cases, the detector array may include detectors capable of detecting the arrival times of individual ions or ion clusters.

In some embodiments, one or more detectors may be located further downstream of the mass filter. The detector may comprise any suitable detector capable of detecting ions or clusters of ions. If there is more than one detector, these detectors may each independently be the same or different. Examples of specific detectors include, but are not limited to, faraday cups, electron multipliers, dynodes, charge Coupled Devices (CCDs), CMOS sensors, fluorescent screens, and the like.

As described above, in some embodiments, the mass spectrometer comprises one detector or an array of detectors. In some embodiments, the detector array includes a channel electron multiplier (e.g.Detector) and/or dynodes. Other non-limiting examples of detection include imaging detectors such as micro-channel plate (MCP) arrays, CCDs, and/or CMOS sensors. More than one of these and/or other detectors may be present within an array.

The detector array may include any number of detectors for determining ions and/or ion clusters. For example, in some embodiments, the detector array may include at least 2, at least 3, at least 5, at least 10, at least 20, at least 25, at least 30, at least 40, at least 50, etc. detectors. The detector may be positioned to receive ions or ion clusters deflected by passing through a mass filter (e.g., a magnetic mass filter). For example, ions or ion clusters may be deflected at various angles, and detectors forming an array are positioned to receive such ions or ion clusters that are expected to deflect at different angles. Thus, any suitable number of detectors may be used to determine the mass and/or mass-to-charge ratio of a single emitted ion or ion cluster. In some embodiments, the number of detectors can be related to the number of base components (e.g., monomers such as amino acids, nucleotides) present in the biopolymer.

In some aspects, a system such as described herein may allow a mass spectrometer to have relatively high ion transport efficiency, e.g., ions generated at an ion source are determined using one or more detectors, e.g., within a detector array. Without wishing to be bound by any theory, it is believed that many or even most of the ions generated by the ion source may be efficiently directed at the detector (e.g., using ion optics, magnetic mass filters, etc.) due to the lack of air molecules in the mass spectrometer (e.g., because of a relatively low pressure environment), and/or the use of mass filters that do not lose ions or ion clusters (e.g., as is the case with mass filters that sweep across a narrow mass transmission window), resulting in surprisingly low loss rates. Thus, in some embodiments, a mass spectrometer such as described herein may include a total ion transport efficiency (e.g., a ratio of detected ions and ion clusters to ions and ion clusters exiting from the fluid at the capillary opening) of greater than 0.01, and in some cases, transport is at least 0.02, at least 0.03, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.75, at least 0.8, etc. In some cases, the total ion transport is no more than 1, no more than 0.9, no more than 0.8, no more than 0.75, no more than 0.7, no more than 0.6, no more than 0.5, no more than 0.4, no more than 0.3, no more than 0.2, no more than 0.15, no more than 0.1, no more than 0.05, or no more than 0.02. Combinations of the above ranges are possible (e.g., at least 0.02 and no more than 0.9, or at least 0.1 and no more than 0.8). Other ranges are also possible. The total ion transport may be measured by measuring the ratio of the current detected at the detector to the ion source emission current.

Accordingly, based on this relatively high ion transport efficiency, in some aspects, certain systems and methods of determining molecular concentration are described herein. The molecule may be any molecule that can be ionized using an ion source, such as discussed herein. In one set of embodiments, the molecules are dissolved in the fluid. As described above, the molecules may be ionized as ions or clusters of ions from the fluid, after which the ions or clusters of ions may pass through a magnetic mass filter and be directed to a detector array as described herein. Examples of such molecules include, but are not limited to, monomers or biological monomers (amino acids, nucleotides, etc.), such as those described herein. Furthermore, it should be appreciated that the molecules detected in this manner may be ionized to form more than one ion or ion cluster, e.g., such that the concentration of the ion or ion cluster may be used to determine the concentration of the starting molecule.

Further, in one set of embodiments, the fluid may include one or more types of molecules. In some embodiments, the relative amounts (e.g., concentrations) of one or more types of molecules may also be determined by determining the identity and/or time of arrival of one or more types of molecules.

In some aspects, ions or ion clusters that reach the detector may be detected with relatively high temporal resolution. As described above, the time of ions or ion clusters generated at the ion source may be determined with relatively high resolution, and such ions or ion clusters may be directed toward the detector array, such as discussed herein, without substantial obstructions such as air molecules, narrow mass transfer windows, etc., which may make it difficult to determine the timing and/or path of movement of ions or ion clusters from the ion source to the detector. Thus, in some cases, such ions and/or ion clusters may be determined, for example, with a temporal resolution of better than 100 nanoseconds (e.g., better than 75 nanoseconds, better than 50 nanoseconds, better than 25 nanoseconds, better than 10 nanoseconds, etc.). In one set of embodiments, the time resolution may be determined between two (or more) ions or ion clusters striking different detectors in the array.

Accordingly, in some embodiments, a method of determining a duration is described herein. For example, the duration between the time that an ion or ion cluster leaves the ion source and the time that the ion or ion cluster reaches the detector may be determined. In some embodiments, the duration may be determined by monitoring the time that the ion or ion cluster reaches the detector and the time that the ion is emitted from the ion source. Further, in some embodiments, the ion detection rate of the detector is greater than or equal to the rate at which the ion source generates (i.e., emits) ions or ion clusters. In some cases, the detector is capable of detecting each emitted ion or ion cluster that reaches the detector.

In some embodiments, the duration between the time the ion or ion cluster leaves the ion source and the time the ion or ion cluster reaches the detector is greater than or equal to 10 microseconds, greater than or equal to 25 microseconds, greater than or equal to 50 microseconds, greater than or equal to 75 microseconds, and greater than or equal to 100 microseconds. In some embodiments, less than or equal to 100 microseconds, less than or equal to 75 microseconds, less than or equal to 50 microseconds, less than or equal to 25 microseconds, less than or equal to 10 microseconds. Combinations of the above ranges are possible (e.g., greater than or equal to 10 microseconds and less than or equal to 100 microseconds). Other ranges are also possible.

The duration between the time the ion or ion cluster leaves the ion source and the time the ion or ion cluster reaches the detector can be determined with a relatively high time resolution. For example, the duration may be determined with a time resolution of better than 1 microsecond, better than 500 nanoseconds, better than 250 nanoseconds, better than 100 nanoseconds, better than 50 nanoseconds, better than 10 nanoseconds, better than 5 nanoseconds, and so on.

Certain aspects relate to sequencing polymers, such as biopolymers, using an instrument that includes an ion source, such as a mass spectrometer as described herein. For example, in some embodiments, the polymer may be a species of interest. The species of interest may be a biopolymer, such as a protein or peptide (including amino acids), or a nucleic acid sequence (e.g., DNA, RNA, etc.). Other types of biopolymers, such as carbohydrates or polysaccharides, may also be used as the species of interest in some cases. Furthermore, it should be understood that in some cases other types of polymers may also be sequenced, such as synthetic or artificial polymers. Furthermore, similarly, structures of the species of interest that are not polymers can also be determined.

In some cases, for example, the structure, sequence, and/or identity of the species of interest (e.g., polymer) can be determined by determining the ionization fraction using a detector. For example, the sequence of a species of interest may be detected by monitoring the time that a single ionised fragment (e.g. ion or ion cluster) reaches the detector, e.g. by ionising a polymer as described above and generating ions or ion clusters. Without wishing to be bound by any theory, it is believed that the species of interest, such as a polymer, may be ionized in a substantially linear manner, e.g., due to the size of the capillary opening, and the ions or ion clusters generated may then be determined by the detector discussed herein, e.g., in the order in which ions or ion clusters are generated from the species of interest. In some embodiments, the capillary comprises carbon nanotubes or boron nitride nanotubes, wherein the cross-sectional dimension (e.g., inner diameter) of the nanotubes is sufficiently small, e.g., 1nm to 2nm, such that the polymer molecules can reflect sequential ionization of the primary structure of the polymer. Of course, in other embodiments, larger diameters or other materials are also possible, such as discussed herein. It should be noted that in some cases, such as when ions or ion clusters enter a reduced pressure environment, the detector may be able to determine such ordering with relatively high fidelity, for example, because there is relatively little collision with gas molecules as the ions or ion clusters pass through the detector. Thus, based on determining the order of ions or ion clusters, the structure or sequence of the species of interest can be determined.

In some embodiments, methods of sequencing a species of interest are described herein. In one set of embodiments, the sequence of the species of interest (e.g., biopolymer) is determined by determining ions or ion clusters with a detector array. As described above, molecules in a species of interest (e.g., a biopolymer) may be ionized using an ion source to produce an ion or ion cluster (e.g., an ion with a solvent molecule) sequence. In some cases, the ion source may produce predominantly single ions, with few, if any, ion clusters.

In some embodiments, ions or ion cluster (e.g., single ion) sequences emitted from the ion source may substantially retain the biopolymer sequences prior to ionization. For example, at least 50% (at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, etc.) of the ions or clusters of ions are sequentially emitted. Ions or ion clusters may be directed to a detector array as the ions or ion cluster sequences pass through a mass filter, such as a magnetic mass filter. Thus, in certain embodiments, the detector array may be used to determine the mass-to-charge ratio of an ion or ion cluster and/or the arrival time (e.g., time at detection) of an ion or ion cluster.

Advantageously, according to some embodiments, the arrival time of an ion or ion cluster may provide information about the ion or ion cluster sequence at the time of detection. In some cases, a majority of the ions or ion clusters that reach the detector array (e.g., a single ion detector) arrive in a sequence, e.g., the same sequence of emitted ions or ion clusters from the ion source. For example, at least 50% (at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, etc.) of ions or ion clusters arrive in sequence at the detector array. Thus, in some embodiments, the detector array may be used to determine the identity of ions or ion clusters and/or the order of ions or ion clusters. In some cases, the sequence of the species of interest (e.g., biopolymer) may be determined based on the sequence of the ions or ion clusters.

Some aspects are directed to mass spectrometers comprising an ion source as described herein. In some cases, a mass spectrometer may include individual components, such as a vacuum chamber (e.g., capable of producing any reduced pressure described herein), ion optics (e.g., one or more lenses, such as a single lens, etc.), a mass filter (e.g., a quadrupole mass filter, a magnetic sector mass filter, etc.), a detector, an ion bender, an ion trap, etc., in addition to an ion source such as described herein. These and other components will be discussed in more detail herein.

For example, in one set of embodiments, a mass spectrometer or other device as discussed herein may include an ion source having a capillary as disclosed herein. The device may also have an electrode near the capillary. For example, certain embodiments relate to an ion source that includes a capillary defining an opening and an electrode positioned adjacent the opening. The capillary tube may have an opening at the end or tip of the capillary tube. The opening may have any of a variety of cross-sectional dimensions and may be of any shape, such as circular, oval, square, etc. In some embodiments, the opening comprises a cross-sectional dimension of less than 150nm, less than 130nm, 125nm, less than 120nm, less than 110nm, less than 100nm, less than 90nm, less than 80nm, less than 75nm, less than 70nm, less than 65nm, less than 60nm, less than 55nm, less than 50nm, less than 45nm, less than 40nm, less than 35nm, less than 30nm, less than 25nm, less than 20nm, less than 15nm, less than 10nm, less than 5nm, less than 2nm, and the like. Further, in some cases, the opening may have a cross-sectional dimension of at least 1nm, at least 5nm, at least 10nm, at least 15nm, at least 20nm, at least 25nm, at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, etc. Combinations of these are also possible; for example, the cross-sectional dimension of the opening may be between 50nm and 100 nm. While the above embodiments describe a capillary tube having an opening at the end or tip of the capillary tube, it should be understood that not all embodiments described herein are so limited, and in some embodiments the capillary tube may additionally or alternatively have multiple openings along the sides of the capillary tube. Further, in some cases, the device may have one or more holes or openings, such as in a channel or other structure. Thus, the opening need not be an opening of a capillary tube.

In some embodiments, the capillary tapers at the opening. For example, the capillary tube may have a constant taper, e.g., such that the tip of the capillary tube is tapered. Any suitable angle may be present. For example, the angle may be less than 15 degrees, less than 10 degrees, less than 9 degrees, less than 8 degrees, less than 7 degrees, less than 6 degrees, less than 5 degrees, less than 4 degrees, less than 3 degrees, less than 2 degrees, or less than 1 degree (where 0 degrees means no taper, i.e., the capillary is cylindrical). Further, in some cases, the angle of the taper may be at least 1 degree, at least 3 degrees, at least 5 degrees, etc., in some cases. Combinations of these ranges are also possible, for example the taper may be between 1 degree and 5 degrees.

In some embodiments where the capillary tube is tapered at the opening, a laser pulling technique may be used to fabricate the tapered opening. It should be understood that techniques other than laser drawing techniques may be used to produce capillaries with tapered openings. It should also be appreciated that while the capillary tube discussed herein has a tapered opening, in other examples, the opening of the capillary tube may be non-tapered.

In certain embodiments, the capillary of the ion source comprises quartz. Other examples of materials that may be used to fabricate the capillary include, but are not limited to, glass (e.g., borosilicate glass), plastic, metal, ceramic, semiconductor, carbon nanotubes, boron nitride nanotubes, and the like.

In some embodiments, the capillary has a relatively high aspect ratio, such as the ratio of the length of the capillary to the cross-sectional dimension (e.g., diameter) of the capillary opening. For example, the capillary tube may have an aspect ratio greater than 10000. However, it should be understood that the aspect ratio is not limited thereto. For example, in some examples, the aspect ratio of the capillary length to the opening cross-sectional dimension may be greater than 10, greater than 100, greater than 1000, greater than 10000, greater than 100000, or greater than 1000000.

The capillary tube may have a circular or non-circular cross-section (e.g., square). Furthermore, in some embodiments, the capillary tube may have a relatively small cross-section, such as a diameter. For example, the cross-sectional dimensions of the capillary tube may be less than 200nm, less than 150nm, less than 100nm, less than 75nm, less than 60nm, less than 50nm.

Some embodiments of the ion source further comprise an electrode positioned adjacent to the capillary, such as an opening of the capillary. The electrodes may be used to apply an electric field (e.g. as described below) to the fluid within the capillary, for example to the meniscus. In some cases, the fluid within the capillary may be in contact with the counter electrode, e.g., such that a voltage difference between the electrode near the capillary opening and the counter electrode within the capillary can create an electric field for the fluid. In some embodiments, the electrodes may be positioned to produce an electric field maximum near the capillary opening. For example, in some embodiments, the electrodes may be located within 50mm, within 40mm, within 30mm, within 20mm, within 15mm, within 10mm, within 5mm, within 3mm, within 2mm, within 1mm, etc. of the capillary opening.

In some embodiments, the electrode may be located around the capillary, or may be located in front of the capillary, for example in front of the capillary opening, or in a downstream direction.

The electrodes may have any suitable shape. In some cases, the electrodes are circular or circularly symmetric, or symmetrically placed with respect to the capillary. However, other shapes or arrangements are also possible.

In some embodiments, the electrodes define openings (e.g., holes). Thus, in some cases, the electrode may be annular. The electrode may be positioned such that ions or clusters of ions escaping from the fluid in the capillary pass through the central opening of the electrode. The central opening of the electrode may be any shape including, but not limited to, a circle that may be positioned annularly around the capillary opening. In some cases, the opening may also be non-circular. In some embodiments, the opening of the electrode is positioned coaxially with the opening of the capillary. That is, in some embodiments, the opening may be aligned with the opening of the capillary, e.g., such that an imaginary line passing through the center of the capillary cross-section passes through the central opening of the electrode. This may help to apply an electric field to the fluid in the capillary, for example, to move ions or ion clusters away from the fluid, as described herein.

For example, in some embodiments, the electrode has a central opening with a cross-sectional dimension (e.g., an inner diameter) that is greater than the cross-sectional dimension of the capillary opening, such as at the end or tip of the capillary. For example, according to some embodiments, the electrode has a central opening with a cross-sectional dimension (e.g., inner diameter) that is at least 5 times greater than the cross-sectional dimension of the capillary opening. However, it should be understood that the ratio of the cross-sectional dimensions of the electrode central opening to the capillary opening is not limited. For example, in some examples, the cross-sectional dimension of the electrode central opening may be at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 75 times, or at least 100 times greater than the cross-sectional dimension of the capillary opening. In some cases, the cross-sectional dimension of the electrode opening may be less than 10cm, less than 5cm, less than 3cm, less than 1cm, less than 5mm, less than 3mm, less than 1mm, etc. Furthermore, in some embodiments, the front side of the electrode is located in front of the capillary opening.

Furthermore, the electrodes themselves may be of any shape (e.g., circular or non-circular). The electrodes may have the same or a different shape than their openings (if present). The electrodes may have any suitable cross-sectional dimensions. For example, the electrode may have a cross-sectional dimension of less than 10cm, less than 5cm, less than 3cm, less than 1cm, less than 5mm, less than 3mm, less than 1mm, etc.

In some embodiments, the electrode comprises steel. Other examples include copper, graphite, silver, aluminum, gold, conductive ceramics, and the like.

Thus, certain embodiments are directed to electrodes capable of generating an electric field. As described above, in some cases, the electrodes may be positioned to produce a maximum electric field near the capillary opening. In some embodiments, the fluid is contained in the capillary tube such that when an electric field is applied by an electrode near the capillary tube opening, molecules within the fluid can ionize and exit from the capillary tube opening, for example, as ions or clusters of ions, as described herein. In some cases, for example, the electrodes and capillary (e.g., the interior of the capillary) may be connected to a voltage source, such as discussed herein.

Thus, in certain embodiments, a voltage source in combination with an electrode may be used to generate an electric field to cause ions or ion clusters to leave the fluid in the capillary, for example as described herein. In some embodiments, a voltage is applied to generate an electric field at least sufficient to ionize molecules within the fluid at the capillary openings, such as to generate ions or ion clusters. For example, in some embodiments, voltages in the range of 80V to 400V may be used to generate the electric field. In some cases, the voltage may be at least 40V, at least 60V, at least 80V, at least 100V, at least 120V, at least 140V, at least 160V, at least 180V, at least 200V, at least 220V, at least 240V, at least 260V, at least 280V, at least 300V, at least 320V, at least 340V, at least 360V, at least 380V, at least 400V, at least 450V, at least 500V, at least 600V, etc. Further, in some cases, the voltage may be no more than 600V, no more than 500V, no more than 450V, no more than 400V, no more than 380V, no more than 360V, no more than 340V, no more than 320V, no more than 300V, no more than 280V, no more than 260V, no more than 240V, no more than 220V, no more than 200V, no more than 180V, no more than 160V, no more than 140V, no more than 120V, no more than 100V, no more than 80V, no more than 60V, and so forth. In some cases, a combination of these voltages is possible. For example, the voltage may be applied between 80V and 360V, etc. In some cases, the voltage may be applied as a constant voltage or a varying or periodic voltage.

As described above, a voltage may be applied to create an electric field maximum near the capillary opening or at the fluid within the capillary (e.g., at the meniscus at the opening). For example, a voltage may be applied to produce an electric field maximum of at least 0.5V/nm, at least 0.7V/nm, at least 1V/nm, at least 1.1V/nm, at least 1.3V/nm, at least 1.5V/nm, at least 2V/nm, at least 2.5V/nm, at least 3V/nm, at least 3.5V/nm, at least 4V/nm, etc. In some embodiments, the electric field maximum may be no more than 5V/nm, no more than 4.5V/nm, no more than 4V/nm, no more than 3.5V/nm, no more than 3V/nm, no more than 2.5V/nm, no more than 2V/nm, no more than 1.5V/nm, no more than 1V/nm. Combinations of these ranges are also possible in some embodiments; for example, the electric field may be between 1.5V/nm and 3.0V/nm, between 1.5V/nm and 4.0V/nm, etc.

Without wishing to be bound by any theory, it is believed that in certain embodiments, when an electric field is applied, the fluid within the capillary forms a charged meniscus and species leave the charged meniscus, for example as ions or clusters of ions. In some cases, the size of the opening of the capillary may be such that at least 10% of the exiting species exits by ion evaporation, for example as ions or clusters of ions. In some cases, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, etc. of the exiting species exits by ion evaporation.

As previously described, according to some embodiments, a tapered charged fluid meniscus may be generated at the opening of the capillary under an electric field. In some embodiments, the tapered fluid meniscus acts as a point source to allow species to exit as ions or clusters of ions.

The fluid meniscus may generate the exiting species by mechanisms such as charged droplets ionized by electrospray and/or ions and ion clusters by ion evaporation. However, in the case of electrospray ionization, the exiting species exiting from the liquid meniscus will exit as a charged droplet containing the exiting species, which will require the presence of a background gas to further break down the droplet into individual ions, typically by a Coulomb fissions (Coulomb fissions) process. Ion evaporation, on the other hand, describes a process in which molecules are ionized directly into ions (e.g., bare ions) or clusters of ions (e.g., ions with solvent molecules) instead of charged droplets. Ion clusters can contain a single ion and multiple solvent molecules, typically in relatively small numbers. For example, an ion cluster may contain no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 solvent molecules.

Thus, for example, in some embodiments, the size of the capillary opening (e.g., the cross-sectional size of the opening is less than 100 nm) is such that charged droplet formation can be avoided and such that at least 50% of the exiting species ionize directly from the conical fluid meniscus at the capillary opening into ions or clusters of ions.

As described above, in some embodiments, capillaries with relatively small openings (e.g., cross-sectional dimensions less than 100 nm) may be associated with the generation of relatively small amounts of solvent molecules in ion clusters, e.g., as described above. In some embodiments, the size of the opening of the capillary (e.g., less than 100 nm) may be such that the plurality of solvent molecules includes less than or equal to a certain amount of solvent molecules, e.g., such that, on average, the ion clusters generated by the ion source contain less than or equal to 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, a plurality (e.g., greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 99%, or all) of the ion clusters comprise one or two solvent molecules.

Furthermore, as discussed, certain embodiments relate to methods of ionizing a fluid using an ion source, such as generating individual ions or clusters of ions. Certain embodiments include delivering a fluid into a capillary that defines an opening having a cross-sectional dimension less than 100nm, or other configurations such as those discussed herein.

In some embodiments, the fluid includes a sample and a solvent. The sample may include any species of interest that may ionize from the capillary opening. For example, according to certain embodiments, the species of interest includes a biopolymer (e.g., a nucleic acid such as DNA or RNA, a peptide or protein, etc.). Other examples include other types of polymers, such as nylon, polyethylene, etc., or other species of interest that are not necessarily polymers, such as biomolecules. Non-limiting examples of biomolecules may include monomers, such as amino acids, nucleotides, and the like. In some cases, the species of interest is unknown, and it is desirable to determine, at least in part, the structure of the species, for example by ionizing the species and detecting ion fragments, such as in mass spectrometry or other related techniques.

In some embodiments, the solvent may be any liquid that can be used to dissolve the sample or species of interest. For example, according to certain embodiments, the solvent comprises water. However, the solvent is not limited to water. In some cases, the solvent may be an aqueous solution, for example having any of a variety of salt concentrations. In some embodiments, the aqueous solution may have the following salt concentrations: greater than or equal to 10mM, greater than or equal to 20mM, greater than or equal to 30mM, greater than or equal to 50mM, greater than or equal to 100mM, greater than or equal to 150mM, greater than or equal to 200mM, greater than or equal to 300mM, greater than or equal to 400mM, greater than or equal to 500mM, greater than or equal to 750mM, greater than or equal to 1M, greater than or equal to 2M, greater than or equal to 5M, or greater than or equal to 7.5M. In some embodiments, the aqueous solution may have the following salt concentrations: less than or equal to 10M, less than or equal to 7.5M, less than or equal to 5M, less than or equal to 2M, less than or equal to 1M, less than or equal to 750mM, less than or equal to 500mM, less than or equal to 400mM, less than or equal to 200mM, less than or equal to 150mM, less than or equal to 100mM, less than or equal to 50mM, less than or equal to 30mM, less than or equal to 20mM, less than or equal to 10mM, etc. Combinations of the above ranges are possible (e.g., greater than or equal to 100mM and less than or equal to 10M, or greater than or equal to 150mM and less than or equal to 1M).

Other examples of solvents that may be used include, but are not limited to, formamide, alcohols (e.g., ethanol, isopropanol, etc.), organic solvents (e.g., toluene, acetonitrile, acetone, hexane, etc.), ionic liquids, inorganic solvents (e.g., ammonia, sulfonyl chloride fluoride, liquid acids and bases, etc.). In some cases, any combination of these and/or other solvents is also possible.

Furthermore, in some embodiments, the fluid includes a solvent (e.g., water) having a relatively high volatility, e.g., to facilitate the generation of ions or ion clusters. For example, water having a boiling point of 100 ℃ is considered to be volatile in some cases. In some embodiments, liquids having boiling points near room temperature may be used to facilitate the generation of ions or ion clusters. In some embodiments, the solvent useful for promoting ion or ion cluster generation may have a boiling point of less than or equal to 100 ℃, less than or equal to 80 ℃, less than or equal to 60 ℃, less than or equal to 40 ℃, less than or equal to 20 ℃, and the like. Further, the boiling point of the solvent may be 10 ℃ or higher, 30 ℃ or higher, 50 ℃ or higher, 70 ℃ or higher, 90 ℃ or higher, or the like. Combinations of these are also possible; for example, the boiling point of the solvent may be between 50 ℃ and 100 ℃. Other examples of solvents having relatively high volatility include, but are not limited to, acetone, isopropanol, hexane, and the like.

In some embodiments, the temperature of the capillary (in addition to the type of fluid it contains) can be varied to control the number of solvent molecules in the resulting ion clusters. In some embodiments, the temperature of the capillary is set such that the plurality of solvent molecules includes less than or equal to a number of solvent molecules, e.g., such that, on average, the ion clusters generated by the ion source contain less than or equal to 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, the temperature is at least 20 ℃, at least 30 ℃, at least 40 ℃, at least 50 ℃, at least 60 ℃, or at least 70 ℃. In some embodiments, the temperature is no more than 80 ℃, no more than 70 ℃, no more than 60 ℃, no more than 50 ℃, no more than 40 ℃, no more than 30 ℃. Combinations of the above ranges are possible (e.g., greater than or equal to 20 ℃ and less than or equal to 80 ℃). In some cases, the temperature of the capillary is controlled by a resistive heater, a Peltier junction, an infrared heater, or the like.

In some embodiments, an appropriate range of electric fields and an appropriate range of capillary opening sizes may be selected to cause at least some molecules to leave as ions or clusters of ions, e.g., as described herein.

In certain embodiments, the ion sources described herein may be used with liquid chromatography mass spectrometry systems. For example, a liquid chromatograph may be coupled to an ion source to separate peptides or other molecules before they are ionized and transported to a mass spectrometer. In some cases, mass spectrometers can be used to perform single or tandem (MS/MS) analysis to identify ionized peptides or molecules, as in proteomic experiments. Advantageously, the use of an ion source (having a capillary with a nano-sized opening and/or tip) as described herein to deliver ions directly into a low pressure environment can increase the sensitivity of the instrument, the ion transport efficiency in such systems, and eliminate the need for multiple pumping stages.

In other embodiments, an ion source as described herein may be used as a nanopipette and ion source. For example, the capillaries with nanoscale tips described herein (e.g., pull quartz capillaries) can be used to pierce cells or tissue and extract their biomolecular content. The capillary can then be inserted directly into a vacuum chamber and the extracted molecules can be ionized and transported to a mass spectrometer. Such techniques may be used, for example, to sample relatively small volumes of liquid, such as the contents of individual cells. For example, this technique can be used in single cell proteomics research.

According to certain aspects, various ion optics may be located downstream of the ion source in addition to the ion source, such that in certain cases, the exiting molecules (e.g., ions and ion clusters) may be transported along a path downstream of the ion source, i.e., the downstream direction is the direction in which the ions or ion clusters travel. In some embodiments, the ion optics include one or more single lenses (e.g., a first single lens and a second single lens). Those of ordinary skill in the art will be familiar with the various ion optics used in mass spectrometry.

Certain aspects include delivering ionized molecules from a fluid directly into a reduced pressure or vacuum environment. Without wishing to be bound by any theory, it is noted that techniques such as electrospray ionization generally require the presence of a background gas to further break up the droplets into individual ions, typically by a coulomb fission process. In contrast, according to certain embodiments, ions or ion clusters generated as described herein may enter such an environment directly without the need for a significant amount of background gas. Thus, certain techniques, such as mass spectrometry, may be performed using a reduced pressure or vacuum environment without necessarily requiring the addition of a background gas.

Thus, in one set of embodiments, the capillary may be positioned to allow ions or ion clusters exiting the opening to enter a reduced pressure or vacuum environment. In some cases, the environment may be an environment having a pressure of no more than 100mPa. In certain embodiments, the environment may have a pressure of no more than 1000mPa, no more than 10mPa, no more than 1mPa, no more than 0.1mPa, etc. In some embodiments, ions or clusters of ions from the fluid are directed into the vacuum environment.

It should be appreciated that some embodiments provided herein focus on delivering ionized molecules directly from a fluid into an environment at a pressure of no more than 100mPa. However, it should be understood that the pressure within the environment is not limited to 100mPa. In some embodiments, the pressure may also be greater than or equal to 100mPa and less than or equal to 1Pa.

As previously described, in some embodiments, the mass spectrometer comprises a pump. The pump may be used to create a reduced pressure or vacuum environment, for example as described herein. Non-limiting examples of pumps include diffusion pumps, molecular drag pumps, turbo-molecular pumps, and the like.

In some embodiments, there may be a relatively high pressure differential between the vacuum chamber and the fluid at the capillary opening. For example, where the fluid enters the capillary, the pressure may be about 1 atmosphere, while inside the vacuum chamber where the opening of the capillary is located, the pressure may be about 100mPa, or other reduced pressures, such as those described herein. However, in some cases such as those described herein, the fluid meniscus at the capillary opening may be relatively stable despite a relatively high pressure differential, for example due to the surface tension of the fluid at the meniscus. For example, the pressure differential across the fluid meniscus at the capillary opening can be at least 0.1 atmosphere, at least 0.2 atmosphere, at least 0.3 atmosphere, at least 0.4 atmosphere, at least 0.5 atmosphere, at least 0.6 atmosphere, at least 0.7 atmosphere, at least 0.8 atmosphere, at least 0.9 atmosphere, at least 1 atmosphere, and the like. Furthermore, in some embodiments, the hydraulic resistance of a fluid in a capillary (e.g., a capillary with an opening less than 100 nm) such as described herein may be higher than the hydraulic resistance in an ion source employed in electrospray ionization.

According to certain embodiments, the capillary openings are sized such that solvents having relatively higher volatility remain unfrozen at the capillary openings when exposed to relatively lower pressures. In some embodiments, the openings of the capillaries are small enough that the relatively high volatility solvent remains unfrozen when entering the surrounding environment. In some embodiments, the opening of the capillary is small enough that the fluid containing the sample and solvent remains unfrozen as the species of interest ionizes, such that at least some of the species of interest ionizes to form ions (e.g., single ions) or clusters of ions.

U.S. provisional patent application Ser. No. 63/015407, entitled "Nanotip Ion Sources and Methods," filed 24, 4/2020, by Stein et al, is incorporated herein by reference in its entirety. In addition, international patent application Ser. No. PCT/US2021/028954, entitled "Nanotip Ion Sources and Methods," filed by Stein et al at month 4, 23 of 2021, is also incorporated herein by reference in its entirety. In addition, U.S. provisional patent application Ser. No. 63/179046, entitled "Systems and Methods for Single-Ion Mass Spectrometry with Temporal Information," filed by Stein et al at 2021, 4, 23, is also incorporated herein by reference in its entirety.

The following examples are intended to illustrate certain embodiments of the present disclosure, but do not illustrate the full scope of the present disclosure.

Example 1

Certain systems and methods are disclosed in the examples below that allow for measurement of a sample (e.g., an amino acid) that is directly emitted into a vacuum (i.e., a reduced pressure environment) by ion evaporation from a surface of a fluid (e.g., an aqueous solution in this embodiment). The systems and methods in these examples are applicable to fluids having relatively low conductivities, e.g., equivalent to about 10mM NaCl. In some embodiments, the method also produces predominantly bare ions or ion clusters having only one or two water molecules. One feature is directed to the nanoscale dimensions (< 100 nm) of the ion source. The small size of the capillary tip creates significant field enhancement and limits fluid flow such that ions from the sample (e.g., amino acids) are ejected directly into vacuum by ion evaporation, rather than from droplet formation and a series of coulomb fissions. Small tip openings (sometimes also referred to as "nanopores") can also prevent large amounts of solvent from evaporating into the vacuum chamber while preventing liquids from freezing, allowing for the investigation of samples of amino acids and the like in volatile solvents such as water.

This example shows various parts of a mass spectrometer for performing experiments according to an embodiment. The experiments described in the examples below were all performed in custom-built instruments called "nano Kong Zhipu instruments" as shown in fig. 1A-1B.

One component of the instrument used in this example is an ion source. The ion source comprises a capillary having an internal tip diameter of less than 100nm and a ring electrode positioned within the system in front of the capillary tip. In this example, the capillary is made of quartz, but in other embodiments the capillary may also be made of borosilicate glass, plastic, metal, ceramic, semiconductor, or other materials. In this example, the diameter of the capillary tapers in size near the tip such that if the shape of the tip is approximately conical, the opening angle of the cone will be in the range of about 1 degree to 5 degrees. The length of the capillary is much greater than the width of its tip, making it extremely high aspect ratio, typically exceeding 10000. Of course, as previously described, other capillary shapes and/or sizes may be used in other embodiments.

In this example, the electrodes are used to induce an electric field at the opening of the capillary tip that is sufficiently high to allow ions to exit directly from the meniscus of fluid generated there, at least in part, by ion evaporation. In this instrument, the electrodes are made of steel, but may also be made of other conductive materials. In this example, the electrode features an aperture through which ions may pass, for example by ion evaporation, exiting from the meniscus of the fluid. In this instrument, the electrode has the shape of a washer, i.e. a disc with a circular hole in the middle, although other shapes may be used. In this instrument, the diameter of the intermediate hole is about 1cm, but this size is not critical. For example, it may be at least 10 times larger than the capillary tip diameter or other dimensions described herein. The outer diameter of the electrode is about 5cm, but this dimension is not critical either. It may be larger than the bore diameter. In this experiment, the front side of the electrode defines a plane behind which the tip of the capillary is located, with the distance being in the range of about 1mm to 5mm, with the capillary axis aligned with the electrode axis.

A voltage, typically in the range 80V-400V, is applied between the fluid and the electrode to cause ions to leave the fluid. The Ag/AgCl wire in the capillary acts as a counter electrode. Two single lenses are used to focus the outgoing ion beam through the aperture in the electrode. Ions are analyzed by a quadrupole mass filter in the instrument, but different types of mass filters, such as magnetic sectors, may also be used.

Example 2

This example illustrates a mass spectrometer according to some embodiments that is capable of determining the mass-to-charge ratio (m/z) of ions emitted from a nanopore ion source at any time and the precise moment of detection. Knowledge of the relative timing of ion detection allows for the determination of ion association, ion ordering and ion sequence. The instrument in this example achieves the ability to provide time information by combining a magnetic mass filter with a single ion detector array. This example demonstrates that this instrument is sensitive to multiple different amino acids arriving simultaneously and has a temporal resolution of less than 1 microsecond.

The nanopore ion source may deliver individual amino acid ions directly from the formamide or aqueous solution to the high vacuum portion of the mass spectrometer. See U.S. patent application Ser. No. 63/015407, entitled "Nanotip Ion Sources and Methods," issued 24, U.S. Pat. No. 4,2020, to Stein et al, and PCT application entitled "Nanotip Ion Sources and Methods," issued 23, U.S. Pat. No. 4,2021, each of which is incorporated herein by reference in its entirety. For example, figure 2 shows a mass spectrum of 14 different amino acids delivered from an aqueous solution through a capillary tip having a diameter of less than 100 nm. Notably, the amino acid ions are predominantly naked, not aggregated with solvent molecules; this makes interpretation of the data relatively simple. It is also notable that these high mass spectra were obtained from very small ion emission currents (-10 pA) and using very low extraction voltages (-200V).

In fig. 2, a mass spectrum of positive amino acid ions delivered directly from a nanopore ion source into a high vacuum is shown. The amino acid is dissolved in an aqueous solution. In each case, the pH is adjusted with acetic acid to below the isoelectric point of the amino acid in solution.

In this example, an instrument capable of determining a single ion was designed and manufactured. This is schematically shown in fig. 3. The instrument incorporates a nanopore ion source, a magnetic mass filter, and a single ion detector array. The magnetic mass filter is made of neodymium magnets and ferromagnetic yokes. The mass filter produced a cylindrical region of approximately 5cm in diameter and approximately 1cm in height, in which region a magnetic field oriented in the axial direction was present. The magnetic field measurement was about 0.6T. Ions pass horizontally through the cylindrical region (perpendicular to the cylindrical axis) and are magnetically affected, causing the ions to fan out according to their m/z. A single ion detector array receives the fan-shaped ions and determines the m/z of each ion based on the impact location.

The ion detector array may includeA detector (e.g., an electron multiplier) and dynodes. The detector array may also include an imaging detector, such as a microchannel plate (MCP) array, a CCD or CMOS sensor. Because the instrument in this example determines the mass of ions based on the location at which the ions strike the detector array, the time domain can be used to establish the order of ion emission. Thus, the instrument can measure amino acid sequences by interpreting the sequence of pulses of the detector array. / >

Example 3

This example describes a technique for sequencing a single protein. Single protein sequencing methods based on fluorescence sequencing, nanopore and tunneling spectroscopy are under development and show promise. However, only Mass Spectrometry (MS) has demonstrated the ability to identify amino acids with minimal degeneracy. Existing MS ion sources have low ion transfer efficiency and disrupt the spatial ordering of ions. An ion source is provided herein that includes a glass capillary having pores with diameters less than 100nm that emit amino acid ions directly into a high vacuum from an aqueous solution. The individual ions travel in a collision-free trajectory before striking the individual ion detector. In this example, unsolvated ions of 16 different amino acids were measured, as well as glutathione and its two post-translational modified variants. This example discusses a method of sequencing a single protein based on MS and a nano-capillary ion source.

Mass spectrometry has been the dominant source of proteomics research for decades; its utility derives from the ability to distinguish amino acids by mass and the availability of fragmentation techniques that can probe protein structures in tandem MS (MS/MS) measurements. Furthermore, the development of soft ionization techniques, particularly electrospray ionization (ESI), is critical for the complete transfer of peptide ions into the gas phase. ESI, however, suffers from low ion transfer efficiency, limiting the sensitivity of mass spectrometry; millions to billions of copies of protein are required to reach the detection limit of a typical instrument.

As shown in fig. 4A, ESI delivers analytes from a stream of charged droplets present in an electrically-induced liquid cone-shaped jet at the end of a capillary to a mass spectrometer. Each droplet carrying a large number of charges and analyte molecules must undergo a series of evaporation and coulomb explosion cycles before the ions can be used for gas phase analysis. Only a small fraction of the analyte molecules eventually appear as gas-phase ions and of those analyte molecules that appear, a large fraction collides with the wall of the transfer capillary before entering the low-pressure region where the mass filter and detector are located. ESI typically transfers only 1/106 of the ions to the mass filter. The nano electrospray ionization (nano-ESI) technique improves the transport efficiency to 0.1% -1% by using smaller capillaries (about 1 micron tip diameter). Hydraulic ion focusing is another technique that significantly improves transmission efficiency with optimized pore size between vacuum stages. However, even though transfer efficiencies are nearly uniform, all of the above techniques face separate challenges of sequencing. The use of atmospheric background gas to facilitate desolvation of ions creates an environment in which the mean free path of amino acids is less than 50nm and collisions will rapidly disrupt the spatial ordering required for single protein sequencing.

Described in this example is an ion source that emits amino acid ions directly into a high vacuum (fig. 4B). The core of the ion source is a drawn quartz capillary tube with a tip diameter of less than 100nm. The small tip can affect emission in various ways: first, the fluid flow may be about three orders of magnitude lower than in nano-ESI and lower than the minimum flow required to form a stable cone jet. Without the conical jet, the formation of charge droplets may be prevented. In some cases, the surface tension of the water meniscus stretched over the nanoscale opening can support many atmospheres and maintain a stable liquid-vacuum interface. Furthermore, in some cases, the electric field is concentrated at a sharp conductive tip, just like a nano-capillary filled with electrolyte. As a result, under certain conditions, an electric field of 1V/nm can be obtained at the liquid meniscus. At this high field strength, ions will escape from the liquid at a high rate through the ion evaporation process.

An overview of the mass spectrometer described in this example is shown in fig. 4C. By Ag/AgCl electricity inside the nano-capillaryA voltage is applied between the pole and an annular extraction electrode located about 5mm in front of the tip of the nano-capillary, emitting ions from the source. The ions pass through focusing ion optics, a quadrupole mass filter (Extrel) and an ion bender before being measured by a continuous dynode single ion detector. The background pressure inside the instrument is typically 10 ^-6 The average free path of amino acid>10 m) is more than an order of magnitude larger than the size of the instrument.

An aqueous solution of 16 different amino acids, except tryptophan, was prepared at a concentration of 100mM, and at a concentration of 50mM due to its lower solubility. To produce positive amino acid ions, the pH of each solution was lowered below the corresponding isoelectric point by the addition of acetic acid. The nano-capillaries were pre-filled with an amino acid solution prior to insertion into the vacuum chamber. Applying an extraction voltage V in the range of +260V to +360V between the nano-capillary and the extraction electrode _e An emission of an ion current of several picoamps is induced (fig. 4B). Attacks typically occur suddenly with ion measurements that strike the instrument detector at a rate sufficient to collect a clear mass spectrum in a few minutes to a few hours.

A mass spectrum of an arginine solution obtained using a nano-capillary ion source with an inner tip diameter of 41nm is shown in fig. 4D. Five peaks are clearly visible. The peak at 174m/z corresponds to singly charged arginine ions (Arg ⁺ ). The higher m/z peaks are all 18m/z apart, and this shift is caused by additional water molecules. Thus, the other peaks correspond to arginine (Arg) ⁺ (H ₂ O) _n ) Wherein the solvation number n ranges from 1 to 4.

Fig. 4A is a schematic diagram of conventional electrospray ionization showing background gas that stimulates evaporation of solvent from the droplet and a transfer capillary where significant ion loss occurs. Fig. 4B is a schematic diagram of a nanocapillary ion source showing a liquid filled nanocapillary tip, an extractor electrode, and Ve applied therebetween. The inset shows an SEM image of a drawn quartz nanocapillary tip with an inner diameter of 30 nm. Fig. 4C is a schematic diagram of a mass spectrometer used in the present study. Ion optics consisting of extractor electrodes and a single lens extract ions from the liquid meniscus at the ion source and focus them through a quadrupole mass filter and an electrostatic ion bender. The transported ions strike a channel electron multiplication detector. FIG. 4D shows a mass spectrum of a 100mM arginine aqueous solution obtained with a 41nm inner diameter nano-capillary ion source in a quadrupole mass spectrometer.

The effect of tip diameter on arginine mass spectrum is shown in figure 5A. The mass spectra shown were obtained using nanocapillary tubes with internal tip diameters of 300nm, 125nm and 20 nm. The largest nanotip produces a broad spectrum of peaks, including bare arginine ions, eight incrementally hydrated arginine ion clusters, and ions corresponding to arginine dimer (Arg Arg+H) ⁺ 349 m/z. A medium-sized tip produces a narrower spectrum including bare arginine ions, six incremental hydrated arginine ion clusters, and a relatively reduced arginine dimer ion peak. The smallest tips produced mainly bare arginine ions, but decaying peaks corresponding to the mono-and di-hydrated arginine ion clusters could also be seen in the spectra. Smaller tips tend to produce a spectrum of relatively stronger signals and less noise than larger tips, as shown by the baselines of the three spectra in fig. 5A. Some variation in solvation state distribution was observed between the nano-capillaries with similar tip sizes (e.g., 20nm as shown in fig. 4D versus 41nm as shown in fig. 5A). However, only nanocapices with inner tip diameters less than about 65nm produce spectra, with most of the amino acid ions being measured in undissolved state.

The nano-capillary source can generate ions of many different amino acids and small peptides for analysis. The solution within the nanocapillary can be conventionally exchanged using a liquid delivery system without interrupting the measurement. Figure 2B shows the mass spectrum of 16 different aqueous amino acid solutions. These measurements used three different nano-capillaries with inner tip diameters of 20, 25 and 60nm, respectively. The most pronounced peak in each spectrum corresponds to singly charged and undissolved amino acid ions. The spectra of glycine, alanine, proline, valine, cysteine, glutamine and phenylalanine show no additional peaks corresponding to solvated amino acid ions. The spectra of serine, threonine, asparagine, lysine, methionine, histidine, arginine and tryptophan show a second peak 18m/z to the right of the undissolved peak, corresponding to the amino acid ion monohydrate. Leucine shows a third and possibly a fourth peak corresponding to a higher solvation state. Tryptophan spectra showed peaks below 200m/z, consistent with the hydration state of hydronium ions, and also appeared in control measurements of aqueous solutions without amino acids. Tryptophan has a lower solubility than the other amino acids studied and produces a relatively weak signal. Four amino acids are missing in this example: aspartic acid and glutamic acid are not included because their low isoelectric points require operation in negative ion mode, isoleucine and leucine are indistinguishable based on m/z, so no study is done and the low solubility of tyrosine results in poor emission characteristics.

The mass spectra of glutathione and two chemically modified variants, s-nitrosoglutathione and s-acetylglutathione, are shown in figure 5C. Glutathione is a tripeptide with biological significance, and the chemical modification forms correspond to common post-translational modification. Each peptide was analyzed in 100mM concentration in water by adding acetic acid to set the pH between 3.1 and 3.9. A nano-capillary having a 20nm inner diameter tip was used to generate ions. Glutathione spectra showed a single peak at 307m/z, which corresponds to undissolved glutathione ions with one charge. The spectra of s-acetyl glutathione and s-nitrosoglutathione show major peaks at 349m/z and 336m/z, respectively, corresponding to singly charged unhydrated peptide ions; the spectrum also shows two progressively smaller peaks 18 and 36m/z to the right of the main peak, corresponding to the mono-and di-hydrated peptide ions, respectively.

FIG. 5A shows the use of a nanocapillary ion source with 3 different inner tip diameters at H ₂ Mass spectrum of 100mM arginine in water in O. Fig. 5B shows a gallery of a 16 amino acid mass spectrum, ordered from top left to bottom right. All experiments were performed using a nano-capillary with an inner tip diameter of 20-60 nm. FIG. 5C shows the overlapping mass spectrum of glutathione and its two PTM variants, s-nitrosoglutathione and s-acetylglutathione.

The conventional electrospray mechanism shown in fig. 4A is excluded as the primary source of measuring ions for two reasons. First, nano-sized droplets in high vacuum shed only a small portion of their mass before freezing due to latent heat loss during evaporation. The instrument described in this example lacks the background gas required to maintain solvent evaporation and ion emission during electrospray. Second, the small size of the nano-capillaries used in this example limit the fluid flow out of the tip to 0.1nL/min or less: at least three orders of magnitude lower than the minimum flow required to maintain a stable cone jet.

Instead, it is believed that ions are emitted directly from the meniscus at the ion source tip by ion evaporation. Fig. 4B is a schematic diagram of pure ion mode emission. The theory of ion emission of taylor cones predicts that pure ion mode dominates when the ratio K/Q of liquid conductivity to flow is large. In liquid metals and ionic liquids, this is achieved by a very high electrical conductivity k. As shown in this example, the same effect is achieved with a very small flow rate Q. The size of the nano-capillary impedes the flow of the liquid, limiting the flow to below 0.1 nL/min. Fig. 6B shows the predicted flow through the nanocapillary presented in this example as a function of tip radius. A stable conical jet cannot be formed due to insufficient flow, resulting in a closed meniscus with high curvature. At such a meniscus, the local electric field may reach a value of greater than or equal to 1V/nm, which is high enough for ion evaporation to occur. FIG. 6C shows as r ₀ The characteristic maximum electric field of the conical jet surface of the function of (2). The field is equal to r ^-1/2 Proportional to r, the field required for ion evaporation ₀ Irrespective of the fact that the first and second parts are. As a result, when r ₀ Very little time, ion evaporation is considered to be the primary ion emission mechanism.

In conjunction with the mechanism of gradual cleavage of amino acids from peptides, single molecule protein sequencing mass spectrometers are envisaged, as shown in fig. 6D. Proteins develop near the tip of the nanocapillary, probably due to interactions between positively charged protein sites and negatively charged silanol groups on the capillary surface. The unfolded protein can be split into individual amino acids or small peptides by photolysis within the nano-capillary ion source and then can be emitted by ion evaporation. The ions are focused by a set of ion optics and pass through a magnetic sector that separates them according to their mass-to-charge ratio. The ions strike an array of electron multiplying detectors, and by taking into account the time and location of each ion detection event, the original sequence can be reconstructed. The 19 detectors can be used to distinguish all amino acids except leucine/isoleucine. Electron multipliers commonly used in mass spectrometry can detect ions at a rate of about 100 MHz.

Fig. 6A is a graph comparing the cone spray mode ion emission of conventional electrospray with the pure ion mode emission from a nanocapillary. Fig. 6B shows a theoretical prediction of flow through a tapered capillary as a function of tip inner diameter using a frustoconical model. Fig. 6C shows a theoretical prediction of the characteristic electric field in the transition region of the cone-shaped jet at the capillary tip as a function of the inner radius of the capillary tip, and compares with the predicted electric field required to achieve evaporation of ions from water. Fig. 6D is a schematic diagram of a contemplated mass spectrometer capable of single molecule protein sequencing. Fig. 6E shows a theoretical calculation of the cumulative probability that an emitted amino acid will collide with an evaporated water molecule or background gas molecule as a function of distance from the meniscus. The dashed line shows the calculated maximum possible water vapor density as a function of distance from the meniscus, calculated using the Hertz-Knudson model with an evaporation coefficient of 1.

The device in this example measured a steady emission current as low as 10pA, corresponding to an ion emission rate of 60 MHz. Thus, the detector speed is sufficient to identify each ion emitted by the nanocapillary ion source. This sequencing strategy relies on sequence preservation in the fragmentation, emission and mass separation stages.

As shown in this example, the nanocapillary ion source is capable of emitting singly charged, undissolved biomolecular ions directly into a high vacuum. 16 of the 20 proteinogenic amino acids, glutathione, s-nitrosoglutathione and s-acetylglutathione were released mainly in undissolved state by mass spectrometry analysis.

Although various embodiments of the disclosure have been described and illustrated herein, various other means and/or structures for performing a function and/or obtaining results and/or one or more advantages described herein will be readily apparent to those of ordinary skill in the art, and each such variation and/or modification is deemed to be within the scope of the disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

In the event that the present specification and documents incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference contain conflicting and/or inconsistent disclosure, documents with a later date of validation will control.

All definitions defined and used herein should be understood to control dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an" as used in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

The phrase "and/or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that in some cases exist in combination and in other cases exist separately. The various elements listed as "and/or" should be interpreted in the same manner, i.e. "one or more" such elements are combined. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether or not related to those elements specifically identified. Thus, as a non-limiting example, in an embodiment, references to "a and/or B" may refer to a alone (optionally including elements other than B) when used in conjunction with an open language such as "include"; in another embodiment, refer to B only (optionally including elements other than a); in yet another embodiment, applies to both a and B (optionally including other elements), and so on.

As used in the specification and claims, "or" should be understood to have the same meaning as "and/or" defined above. For example, when separating items in a list, "or" and/or "should be construed as inclusive, i.e., including at least one, but also including more than one, multiple or series of elements, and optionally additional unlisted items. Only the opposite terms, such as "only one" or "exactly one," or when "consisting of …" is used in the claims, will be referred to as comprising exactly one element of a plurality or series of elements. In general, the term "or" as used herein should be interpreted only as referring to an exclusive choice (i.e., "one or the other, but not two") when preceded by an exclusive term, such as "either," one, "" only one, "or" exactly one.

As used in the specification and claims, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. The definition also allows that elements may optionally be present in addition to elements specifically identified in the list of elements to which the phrase "at least one" refers, whether or not associated with those elements specifically identified. Thus, as a non-limiting example, "at least one of a and B" (or equivalently, "at least one of a or B," or equivalently "at least one of a and/or B") may refer in an embodiment to at least one, optionally including more than one, a, absent B (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, is absent a (and optionally includes elements other than a); in yet another embodiment, at least one, optionally including more than one, a, and at least one, optionally including more than one, B (and optionally including other elements), and the like.

When the word "about" is used herein to refer to a number, it is to be understood that yet another embodiment of the present disclosure includes a number that is not modified by the presence of the word "about.

It should also be understood that in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited, unless clearly indicated to the contrary.

In the claims and in the above description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "containing," "consisting of …," and the like are to be construed as open-ended, i.e., to mean including but not limited to. As described in section 2111.03 of the U.S. patent office patent review program manual, only the transitional phrases "consisting of …" and "consisting essentially of …" are closed or semi-closed transitional phrases, respectively.

Claims

1. A mass spectrometer, comprising:

an ion source comprising a capillary and an electrode adjacent to the capillary, wherein the capillary comprises an opening having a cross-sectional dimension of less than 125 nm;

a magnetic mass filter downstream of the ion source; and

A detector array downstream of the magnetic mass filter.

2. The mass spectrometer of claim 1, further comprising a vacuum chamber housing the ion source.

3. The mass spectrometer of claim 2, wherein the vacuum chamber has a pressure of no more than 100 mPa.

4. A mass spectrometer as claimed in any of claims 2 or 3, wherein the vacuum chamber has a pressure of no more than 10 mPa.

5. The mass spectrometer of any of claims 1-4, wherein the magnetic mass filter comprises a permanent magnet.

6. The mass spectrometer of any of claims 1-5, further comprising ion optics downstream of the ion source and upstream of the magnetic mass filter.

7. The mass spectrometer of any of claims 1-6, further comprising an ion bender configured to deflect ions exiting the mass filter to the detector.

8. The mass spectrometer of claim 7, wherein the ion optics comprise at least one single lens.

9. The mass spectrometer of any of claims 1-8, wherein the mass spectrometer has a time resolution of less than or equal to 1 microsecond.

10. The mass spectrometer of any of claims 1-9, wherein the detector array comprises an electron multiplier.

11. The mass spectrometer of any of claims 1-10, wherein the detector array comprises dynodes.

12. The mass spectrometer of any of claims 1-11, wherein the detector array comprises a microchannel plate array.

13. The mass spectrometer of any of claims 1-12, wherein the detector array comprises a CCD.

14. The mass spectrometer of any of claims 1-13, wherein the detector array comprises a CMOS sensor.

15. The mass spectrometer of any of claims 1-14, wherein the detector array comprises a SQUID.

16. The mass spectrometer of any of claims 1-15, wherein the opening of the capillary has a cross-sectional dimension of less than 65 nm.

17. The mass spectrometer of any of claims 1-16, wherein the opening of the capillary has a cross-sectional dimension of less than 50 nm.

18. The mass spectrometer of any of claims 1-17, wherein the opening of the capillary has a cross-sectional dimension of less than 30 nm.

19. The mass spectrometer of any of claims 1-18, wherein the opening of the capillary tube has a cross-sectional dimension of less than 2 nm.

20. The mass spectrometer of any of claims 1-19, wherein the capillary tube is tapered at the opening.

21. The mass spectrometer of claim 20, wherein the angle of the taper is less than 10 °.

22. The mass spectrometer of any of claims 20 or 21, wherein the angle of taper is less than 5 °.

23. The mass spectrometer of any of claims 1-22, wherein the capillary tube comprises quartz.

24. The mass spectrometer of any of claims 1-23, wherein the capillary tube comprises glass.

25. The mass spectrometer of any of claims 1-24, wherein the capillary tube comprises borosilicate glass.

26. The mass spectrometer of any of claims 1-25, wherein the capillary tube comprises plastic.

27. The mass spectrometer of any of claims 1-26, wherein the capillary tube comprises a metal.

28. The mass spectrometer of any of claims 1-27, wherein the capillary comprises a semiconductor.

29. The mass spectrometer of any of claims 1-28, wherein the capillary comprises carbon nanotubes.

30. The mass spectrometer of any of claims 1-29, wherein the capillary comprises a boron nitride nanotube.

31. The mass spectrometer of any of claims 1-30, wherein an aspect ratio of a length to a cross-sectional dimension of the capillary tube is greater than or equal to 100.

32. The mass spectrometer of any of claims 1-31, wherein an aspect ratio of a length to a cross-sectional dimension of the capillary tube is greater than or equal to 1000.

33. The mass spectrometer of any of claims 1-32, wherein an aspect ratio of a length to a cross-sectional dimension of the capillary tube is greater than or equal to 10000.

34. The mass spectrometer of any of claims 1-33, wherein the capillary tube has a cross-sectional dimension of less than 100 nm.

35. The mass spectrometer of any of claims 1-34, wherein the capillary tube has a cross-sectional dimension of less than 60 nm.

36. The mass spectrometer of any of claims 1-35, wherein the electrode defines a central opening.

37. The mass spectrometer of claim 36, wherein the central opening of the electrode has a cross-sectional dimension of less than 5 cm.

38. The mass spectrometer of any of claims 36 or 37, wherein the central opening of the electrode has a cross-sectional dimension of less than 1 cm.

39. The mass spectrometer of any of claims 36-38, wherein a central opening of the electrode is larger than an opening of the capillary tube.

40. The mass spectrometer of any of claims 36-39, wherein a central opening of the electrode is at least 5 times larger than an opening of the capillary tube.

41. The mass spectrometer of any of claims 36-40, wherein a central opening of the electrode is at least 10 times larger than an opening of the capillary tube.

42. The mass spectrometer of any of claims 1-41, wherein the electrode comprises steel.

43. The mass spectrometer of any of claims 1-42, wherein the electrode is annular.

44. The mass spectrometer of any of claims 1-43, wherein the electrode has a cross-sectional dimension of less than 5 cm.

45. The mass spectrometer of any of claims 1-44, wherein the electrode is located within 10mm of the opening of the capillary tube.

46. The mass spectrometer of any of claims 1-45, wherein the electrode is located within 5mm of the opening of the capillary tube.

47. The mass spectrometer of any of claims 1-46, wherein the electrode is located within 2mm of the opening of the capillary tube.

48. The mass spectrometer of any of claims 1-47, wherein the electrode is located around the capillary tube.

49. The mass spectrometer of any of claims 1-48, wherein the electrode is located in front of the opening of the capillary tube.

50. The mass spectrometer of any of claims 1-49, wherein an imaginary line passing through a center of a cross-section of the capillary passes through a center opening of the electrode.

51. The mass spectrometer of any of claims 1-50, wherein the electrode and the capillary have interiors connected to a voltage source.

52. The mass spectrometer of claim 51, wherein the voltage source is capable of producing a voltage of less than 400V between the electrode and the capillary.

53. The mass spectrometer of any of claims 51 or 52, wherein the voltage source is capable of producing a voltage of less than 360V between the electrode and the capillary.

54. The mass spectrometer of any of claims 51-53, wherein the voltage source is capable of generating a voltage of at least 80V between the electrode and the capillary.

55. The mass spectrometer of any of claims 51-54, wherein the voltage source is capable of generating an electric field between the electrode and the capillary having a maximum value of less than or equal to 4V/nm.

56. The mass spectrometer of any of claims 51-55, wherein the voltage source is capable of generating an electric field between the electrode and the capillary having a maximum value of less than or equal to 3V/nm.

57. The mass spectrometer of any of claims 51-56, wherein the voltage source is capable of generating an electric field between the electrode and the capillary that is at least 1.5V/nm at a maximum.

58. The mass spectrometer of any of claims 1-57, wherein the magnetic mass filter has a magnetic filter strength of at least about 0.5T.

59. The mass spectrometer of any of claims 1-58, wherein the magnetic mass filter comprises a magnet comprising neodymium.

60. The mass spectrometer of any of claims 1-59, wherein the magnetic mass filter comprises a yoke comprising iron.

61. The mass spectrometer of any of claims 1-60, wherein the magnetic mass filter comprises an opening having a first size of at least about 5 cm.

62. The mass spectrometer of any of claims 1-61, wherein the magnetic mass filter comprises an opening having a second size of at least about 1 cm.

63. The mass spectrometer of any of claims 1-62, wherein the detector is a single ion detector.

64. A method of sequencing a biopolymer, comprising:

ionizing a biopolymer contained within a fluid into ions or clusters of ions;

passing ions or ion clusters through a magnetic mass filter;

directing ions or clusters of ions toward a detector array; and

the sequence of the biopolymer is determined by determining ions or ion clusters with a detector array.

65. The method of claim 64, wherein the biopolymer is a protein.

66. The method of claim 65, comprising ionizing amino acids of the protein at a rate of at least 1 amino acid per microsecond.

67. The method of any one of claims 64-66, wherein the biopolymer is a nucleic acid.

68. The method of claim 67, comprising ionizing bases of the nucleic acid at a rate of at least 1 base per microsecond.

69. The method of any one of claims 67 or 68, comprising ionizing bases of the nucleic acid at a rate of at least 10 bases per microsecond.

70. The method of any one of claims 67-69, comprising ionizing bases of the nucleic acid at a rate of at least 100 bases per microsecond.

71. The method of any of claims 64-70, wherein the ions or ion clusters have a total ion transport efficiency of greater than or equal to about 0.8.

72. The method of any of claims 64-71, wherein the ions or ion clusters are generated at a rate of greater than or equal to 1 ion or ion cluster/microsecond to 100 ions or ion clusters/microsecond.

73. The method of any of claims 64-72, wherein a time interval between a molecule exiting near the opening as an ion or ion cluster and an ion or ion cluster being detected at the detector array is greater than or equal to 10 microseconds and less than or equal to 100 microseconds.

74. The method of any of claims 64-73, wherein the detector array comprises an electron multiplier.

75. The method of any of claims 64-74, wherein the detector array comprises dynodes.

76. The method of any one of claims 64-75, wherein the detector array comprises a microchannel plate array.

77. The method of any of claims 64-76, wherein the detector array comprises a CCD.

78. The method of any one of claims 64-77, wherein the detector array comprises a CMOS sensor.

79. The method of any one of claims 64-78, wherein the ions or ion clusters are detectable with a time resolution of better than 100 nanoseconds.

80. The method of any of claims 64-79, wherein ionizing a biopolymer contained within a fluid into ions or clusters of ions comprises ionizing the biopolymer into individual ions.

81. The method of any one of claims 64-80, wherein passing the ions or ion clusters through a magnetic mass filter comprises passing individual ions through a magnetic mass filter.

82. The method of any of claims 64-81, wherein directing the ions or ion clusters toward a detector array comprises directing the individual ions toward a detector array.

83. The method of any of claims 64-82, wherein determining the ions or ion clusters with the detector array comprises determining the single ions with the detector array.

84. A method of sequencing a biopolymer, comprising:

delivering a fluid comprising a biopolymer into a capillary defining an opening;

applying an electric field to ionize the biopolymer near the opening to produce ions or ion clusters;

Directly switching the ions or ion clusters into an environment with a pressure of not more than 100 mPa;

passing the ions or ion clusters through a magnetic mass filter;

directing the ions or clusters of ions toward a detector array; and

determining the sequence of the biopolymer by determining the ions or ion clusters with the detector array.

85. The method of claim 84, wherein the ions or ion clusters have a total ion transport efficiency of greater than or equal to about 0.8.

86. The method of any one of claims 84 or 85 wherein said ions or ion clusters are generated at a rate of greater than or equal to 1 ion or ion cluster per microsecond to 100 ions or ion clusters per microsecond.

87. The method of any one of claims 84-86 wherein a time interval between a molecule exiting near the opening as an ion or ion cluster and an ion or ion cluster being detected at the detector array is greater than or equal to 10 microseconds and less than or equal to 100 microseconds.

88. The method of any of claims 84-87 wherein the detector array comprises an electron multiplier.

89. The method of any of claims 84-88 wherein the detector array comprises dynodes.

90. The method of any one of claims 84-89 wherein the detector array comprises a microchannel plate array.

91. The method of any one of claims 84-90 wherein the detector array comprises a CCD.

92. The method of any one of claims 84-91 wherein the detector array comprises a CMOS sensor.

93. A method of determining concentration, comprising:

ionizing molecules from the fluid into ions or clusters of ions;

passing the ions or ion clusters through a magnetic mass filter;

directing the ions or clusters of ions toward a detector array; and

the concentration of molecules in the fluid is determined by determining the ions or ion clusters with the detector array.

94. A method, comprising:

ionizing molecules from the fluid into ions or clusters of ions;

passing at least 50% of the ions or ion clusters through a magnetic mass filter; and

the ions or clusters of ions are directed to a detector.

95. The method of claim 94, wherein the detector is one detector in a detector array.

96. The method of any one of claims 94 or 95, comprising passing at least 70% of the ions or ion clusters through a magnetic mass filter.

97. The method of any one of claims 94-96, comprising passing at least 80% of the ions or ion clusters through a magnetic mass filter.

98. A method, comprising:

ionizing molecules from the fluid into ions or clusters of ions using an ion source;

passing the ions or ion clusters through a mass filter;

directing the ions or clusters of ions to a detector; and

the duration between the time the ion or ion cluster leaves the ion source and the time the ion or ion cluster reaches the detector is determined.

99. The method of claim 98, comprising determining the duration with a temporal resolution of better than 100 ns.

100. A mass spectrometer, comprising:

an ion source constructed and arranged to generate individual ions or clusters of ions;

a magnetic mass filter positioned to receive individual ions or ion clusters from the ion source;

a pump capable of generating a pressure of less than 100mPa in an environment between the ion source and the magnetic mass filter; and

a detector array positioned to receive the single ion or ion cluster from the magnetic mass filter.

101. A mass spectrometer, comprising:

an ion source;

A magnetic mass filter downstream of the ion source; and

a detector array downstream of the magnetic mass filter.

102. The mass spectrometer of claim 101, wherein the ion source comprises a pulsed laser.

103. The mass spectrometer of any of claims 101 or 102, wherein the ion source comprises a capillary and an electrode in proximity to the capillary, wherein the capillary comprises an opening having a cross-section of less than 125 nm.

104. A method, comprising:

ionizing molecules using an ion source to produce ions or a sequence of ion clusters;

passing the ions or ion cluster sequences through a mass filter; and

the ions or ion cluster sequences are directed to a detector array, wherein at least 90% of the ions or ion clusters that reach the detector array arrive sequentially.